Fig 2: Injection of WT human RERE mRNA results in cyclopia. Injecting increasing concentration of human RERE mRNA results in dose-dependent increase in cyclopia phenotype (A–C). Whole mount in situ hybridization staining of 24 hpf WT and rerea mutant zebrafish embryos with patched2 (D, F) and shha (E, G) probes. Representative images with numbers of embryos showing this pattern are given in each panel.

Fig 3: Morpholino-mediated knockdown of rerea causes coloboma. Increasing concentrations of rerea morpholino was used for knocking down rerea expression (A). Severity of coloboma is denoted by: CI-mild, CII-moderate, CIII-severe. Zebrafish embryos injected with rerea morpholino consistently exhibited coloboma at 48 hpf compared with embryos injected with control morpholino (B). Representative whole mount in situ hybridization staining with pax2a in control and rerea morpholino injected embryos 24 hpf (n ~20 embryos each) (C). WT human RERE mRNA rescues coloboma in rerea morphant fish embryos (D), this rescue was observed to be statistically significant (P < .05) for 20, 40, and 80 pg of mRNA injected, but not for 10 pg. Equal amounts of the human RERE WT and mutant (c.3466 G > A, p.Gly1156Arg) mRNA (40 pg) can rescue coloboma in rerea morphants (P < .05), but the mutant mRNA is not as efficient as WT mRNA (P < .05, E). Student’s t test was used to determine level of significance and “*” denotes significant difference. NS, not significant